Fluorometric assay of primary amines

ABSTRACT

AND ANY OTHER ORGANIC COMPOUNDS CONTAINING A PRIMARY AMINO MOIETY.   A HIGHLY SENSITIVE ASSAY TECHNIQUE FOR PRIMARY AMINECONTAINING COMPOUNDS UTILIZES FLUORESCENCE PRODUCED BY THE INTERACTION OF THE PRIMARY AMINE COMPOUND WITH NINHYDRIN AND AN ARYL ALKYL ALDEHYDE. A PREFERRED ARYL ALKYL ALDEHYDE FOR THE ASSAY TECHNIQUE IS PHENYLACETALDEHYDE. THE ASSAY IS USEFUL FOR THE QUALITATIVE AND QUANTITATIVE DETERMAINATION OF AMINO ACIDS, PEPTIDES, PROTEINS

P 1972 s. UDENFRIEND 3,689,221

FLUOROMETRIC ASSAY OF PRIMARY AMINES Filed 001;. 21, 1970 OPTICAL DENSITY 570 mp. FLUORESCENCE llllllll /OH-LYSINE\ L -LYSINE- ASPARTIC ACID /THREONINE\ O,. O SERINE o 2 GLUTAMIC Ac|D E 8 8 a GLYCINE g ALANINE CYSTINE O VAL|NE/- a -BuFFER o" -METH|ONINE g NORLEUCINE |so| Euc|NE LEucmE TYROS|NE-8 PHENYLALANINE B-ALANINE olz United States Patent US. Cl. 23-230 R 29 Claims ABSTRACT OF THE DISCLOSURE A highly sensitive assay technique for primary amine containing compounds utilizes fluorescence produced by the interaction of the primary amine compound with ninhydrin and an aryl alkyl aldehyde. A preferred aryl alkyl aldehyde for the assay technique is phenylacetaldehyde. The assay is useful for the qualitative and quantitative determination of amino acids, peptides, proteins and any other organic compounds containing a primary amino moiety.

BACKGROUND OF THE INVENTION The use of ninhydrin as a colorimetric reagent for the detection and assay of amino acids, amines and peptides has been known in the art for nearly sixty years. More recently, the ninhydrin color reaction has found extensive application in conjunction with the newly developed amino acid analyzers for the automated microanalysis of amino acid compositions of protein hydrolysates. By the use of micro techniques, it has been possible to apply the colorimetric ninhydrin procedure at the nanomole level. However, this figure represents the limit of sensitivity of the colorimetric technique.

It has also been known in the prior art that ninhydrin can yield highly fluorescent products with amine-containing compounds. For example, McCaman and Robins in their paper in J. Lab. Clin. Med. 59, 885 (1962), disclosed a fluorometric assay technique that utilizes ninhydrin in the determination of serum phenylalanine. Close has reported on a study related to the fluorescence obtained by incubating amino acids or amines with ninhydrin and n-butyraldehyde (see fluorescence assay in Biology in Medicine by S. Udenfriend, Academic Press, New York (1969) at p. 196).

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to an improved assay method for the determination of organic compounds containing a primary amino group utilizing fluorescence techniques. In particular, the invention is concerned with a process which involves reacting an organic compound containing a primary amino group with a certain class of aldehydes and ninhydrin to quantitatively produce a highly fluorescent material which can be detected by standard fluorometric techniques and can yield both qualitative and quantitative data relating to said primary amine. An important feature of this invention is that it makes possible the detection of much smaller quantities of amines, amino acids, and peptides than have heretofore been possible utilizing standard colorimetric and fluorometric techniques.

A major aspect of this invention is the utility for fluorometric assays in solution. In such a process a mixture of the primary amine-containing compound, the aldehyde and ninhydrin in an inert solvent containing a pH buffer is heated and the fluorescence is then measured by standard fluorometric techniques. Quite unexpectedly, it has been found that aryl alkyl aldehydes give complexes that are very highly fluorescent. The aryl alkyl aldehydes comprehended by this invention are defined by the formula Ar(CH CHO 3,689,221 Patented Sept. 5, 1972 ice wherein Ar is an aromatic residue and n is an integer having a value of from 1 to 8. Alde'hydes wherein n has a value of from 1 to 3 are particularly useful. Preferred aryl alkyl aldehydes are those where n is 1 and the aromatic residue is phenyl or alkyl substituted phenyl, e.g. phenylacetaldehyde or 4-methyl phenylacetaldehyde. Phenylacetaldehyde is especially preferred.

For example, under identical reaction conditions, the fluorescence produced by using phenylacetaldehyde as compared with that produced by using n-butyraldehyde, a reagent used in previous fluorometric assays, is at least 3 to -fold greater depending upon the pH. A wide variety of primary amine-containing compounds can beassayed by the present technique. Such classes of compounds include primary amines, e.g. ethylamine, ethanolamine, tryptamine, dopamine, norepinephrine, etc.; atamino acids containing a primary amino group, e.g. glycine, alanine, leucine, valine, etc. and peptides containing a primary amino group e.g. insulin, angiotensin, leucylalanine, etc.

It is important that ammonia, secondary and tertiary amines, and compounds which contain an amino group as part of a larger functionality, such as an amide, guanidine or urethane residue, aiford complexes which have little or no fluorescence and which, thus, do not interfere with the assay for primary amine-containing compounds.

The proportions of the reactants used are not broadly critical but normally it is preferred to use a large excess of the aldehyde and ninhydrin with respect to the amine being determined so that the fluorescent product is quantitatively formed. It is generally preferred to use an excess of ninhydrin as compared with aryl alkyl aldehyde. A ten-fold excess is conveniently utilized.

The incubation temperature is also not critical and can be carried out over a range of from about C. to about the boiling point of the reaction medium. It is preferred, however, to utilize a temperature range of from about C. to about C. The time involved in the incubation varies extensively with the substrate and the pH but is generally in the range of from about 5 minutes to about 4 hours. The pH of the incubation medium is normally between about 4 and 9. However, a pH range of from about 6 to 8 is especially preferable.

The pH is controlled by the addition of a buffer to the reaction medium. Suitable buffers are phosphate buffers and others known in the art. The choice of exact pH for any particular assay is dependent upon many factors such as the substrate and the type of assay being performed. For example, at pH values around 6.0 one observes a higher fluorescence for amines and peptides than for neutral amino acids. Although, in general, the absolute intensity of the fluorescence is slightly lower at the lower pH, it offers considerable specificity for the determination of amines and peptides. As one increases the pH, going for example, from pH 6 to pH 8, the fluorescence values of amino acids are increased relative to that of amines and peptides. Also, as the pH is increased the incubation time can be decreased.

The limits of sensitivity for the assay procedure of the present invention can be determined for a variety of substrates and compared With those for the standard colorimetric ninhydrin assay procedure of Moore and Stein, J. Biol. Chem. 176, 367 (1948). The smallest amount which can be determined by the present method is in the range of 10- to 10- moles; whereas for the colorimetric method l0 moles was the smallest amount detectable. Therefore, the fluorescent ninhydrin aldehyde method of the present invention is at least 10 to times more sensitive than the ninhydrin color reaction.

Fluorometric detection has an inherent advantage over colorimetric detection in that in the former method an absolute quantity is measured, whereas in the latter method 21 diflFerence between two measured values is determined, thus reducing the accuracy and limiting the minimum amount detectable. In essence, the limiting factor for a fluorometric assay is the sensitivity of the detection system, i.e. the instrumentation.

The assay procedure of the present invention has been automated to continuously detect, qualitatively and quantitatively, primary amine-containing compounds in a sample stream. It is particularly useful to employ, as a sample stream, the efiiuent from a chromatographic system, for example, a chromatographic column for the separation of amino acids.

In such a procedure, the sample stream is introduced into the automated analyzer at a constant rate and streams of reagents (ninhydrin, aryl alkyl aldehyde and buffer) are added thereto. A suitable means for controlling the addition of the reagents and the flow rate of the sample stream is by use of a proportionating pump. After the reagents are added to the sample stream, they are thoroughly mixed with the sample stream by, for example, passing through a mixing coil. The mixture is then heated at the appropriate temperature for a predetermined length of time to produce fluorescent substances. The heating is conveniently done by passing the mixture through a coil of fixed length, inserted in a constanttemperature heating bath, so that the residence time in the heated coil is known. The solution, after leaving the heating coil, is cooled and passed into a continuous fiow cell of a fiuorophotometer where it is fluoresced and the fluorescence continuously detected. If desired, the detector of the fluorophotometer may be linked to a suitable recording, data display or print-out device.

Another use for the assay procedure of the present invention is for the qualitative and quantitative determination of primary amine-containing compounds in a mixture 'by a technique involving automated, successive sampling of a multiplicity of samples.

In such a method, a fixed volume of a sample solution is introduced by utilizing, for example, a probe which dips into the sample solution. The sample stream thus introduced is processed as for the automated analyzer described above. The sample solutions are separated from one another within the flowing stream by methods generally used, for example, solvent or gas bubbles. If the bore of the tubing carrying the flowing stream is sufiiciently small, no additional means for separating the sample solutions from one another is necessary, since capillary action will prevent intermixing.

The automated presentation of sample solutions for introduction into the analyzer can be accomplished by techniques generally used in the art.

The automated assay procedure of the present invention can also be used in closed-loop process control systems to monitor the level of primary amine-containing compounds in a reaction medium and automatically control the reaction process by methods such as that described by Hana et al. 'in U.S. Pat. 3,306,096.

Another major use for the present invention is for the detection of primary amine-containing compounds on paper or thin layer chromatograms. In such a system, the sample is applied to the paper or thin layer plate and developed in the normal manner. The paper or plate is then sprayed with a solution containing ninhydrin, the aryl alkyl aldehyde and the pH buffer as described above for the solution assays. The paper or plate is then heated in high yields. The choice of solvent, pH, temperature and time is controlled by the same considerations as discussed above for the solution assays. The techniques used for paper and thin layer chromatograms prior to the detection procedure (e.g., spotting of the sample, development of the chromatogram, etc.) are those normally employed in the art.

The particular advantages of the present invention are enumerated below. First, the sensitivity of this method is at least 10 to times greater than commonly used colorimetric methods and 3 to 30 times greater than fluorometric methods for amine assay. Secondly, this method has a wide scope and has been applied to a large variety of primary amines, amino acids and peptides. A third advantage of the present method is that it is readily suited to automation since the fluorescence is proportional to the concentration of amine substrate over a wide range of concentration. The present technique achieves maximum sensitivity as it starts with a reagent which is not fluorescent but which yields fluorescence only on interaction with primary amine-containing compounds. The fact that ammonia does not react in the present process is of great importance since ammonia is usually present in substantial amounts in protein hydrolysates and it is known that traces of ammonia in the atmosphere can limit the usefulness of known colorimetric and fluorometric methods for amine detection.

Another advantage of this method is that at low pH values, particularly about pH 6, far more fluorescence is obtained with peptides than with amino acids. Peptides, particularly large peptides which yield little color with the classical ninhydrin procedures can be identified by the present method. Comparison of proteins by fingerprinting methods usually requires several milligrams of protein to detect all the peptides. By utilizing the procedure of the present invention one can accomplish the same results using only microgram quantities of the protein. Automated fluorometric assays of peptides obtained from digestion of microgram quantities of proteins make it possible to carry out genetic studies even on the small amounts of protein obtained from a single laboratory animal.

Another important application is in the detection of small amounts of physiologically active peptides in tissues. In addition to the utility for identifying known peptides by assay, the method of the present invention is suitable for the detection of new peptides whose biological roles or activities have not yet been ascertained.

In the examples which follow, all temperatures are expressed in degrees centigrade.

EXAMPLE 1 General procedure for fluorometric assay in solution To a 13 x 100 mm. test tube were added 2.0 ml. of 0.2 M phosphate bufl'er (pH 6, 7, or 8) 0.2 ml. of aqueous ninhydrin (50 mM.), 0.1 ml. of aqueous sample solution (generally 10* to 10- mM.) and finally 0.1 ml. of phenylacetaldehyde in ethanol (10 mM.). After mixing thoroughly, the tubes were covered with aluminum foil and transferred to a 60 water bath for a suitable incubation period; 15 min. at pH 8.0, 60 min. at pH 7.0 and min. at pH 6.0. The tubes were then cooled in an ice bath for a few minutes, kept at room temperature for 10 minutes and fluorescence measured within 1 hour (using an Aminco-Beckman fiuorophotometer with a 1 cm. cell). Excitation was set at 390 m and emission at 490 m '6 EXAMPLE This example illustrates the effect of pH and incubation time on fluorescence.

Relative fluorescence intensity pH 6.0 pH 7.0 pH 8.0

Incubation time (min.) Lys Gly Len-Ala Lys Gly Leu'Ala Lys Gly Len-Ala different ninhydrin concentrations. Incubations were carried out and fluorescene assayed High concentrations ofninhydfin by the procedure of Example 1. Lysine, glycine and Relative m tmole per flask. Len-Ala Ninhydrin Phe-CHgOHO fiuores- 50 2250 200 cence 2O EXAMPLE 6 820 This example illustrates the fluorescence obtained with a the normally occurring amino acids under various pH conditions. A typical dipeptide and ammonia are in- Low concentrations otninhydrin cluded fOI comparison. Len-Ala. Ninliydrin Phe-CHQCHO 50 100 100 Relative fluorescence intensity at pH 1 1 Amino acid 6.0 7. 0 8. 0 1

0 0 0 Fluoroescence 1s presented 1n terms of arbitrary units. 1 2g 21 g The value under each reactant represents m tmoles con- 2 2 18 tained in 2.4 ml. of reaction mixture. The general pro- 2 :34 cedure of Example 1 was used to develop and measure 4 263 399 the fluoroescence. 4 27 4 212 314 EXAMPLE 3 s 351 491 7 282 516 This example illustrates the effect of var1ous aldehydes g 292 349 in producing fluorescence with a peptide and ninhydrin. 3 Egg 2 43 Relative fluorescence at pH Aldehyde 6. O 7. 0 8. 0 Formaldehyde. 11 0 0 Acetaldehydeh 3 7 7 32 5 i ff i 3 146 440 427 SO 11 yra 6 Y e Glutaraldehyde 22 21 0 g 1, g Benzaldehyde 14 0 0 trans-Cinnamaldehyd 13 1 611 0 0 The general procedure of Example 1 was used, employ- 3 ing 10 III/1.1110165 of each amino acid and 120 minutes Twenty ,ul. of each aldehyde in ethanol solution (10 mM.) was added to a mixture containing 40 ,ul. of leucylalaninglycylvaline (1.25 mM.), 75 1.1. of ninhydrin (30 mM.) and 200 l. of 0.6 M phosphate bufler at the pH indicated. After incubating for 90 minutes at 60 the samples were cooled to room temperature and fluorescence was measured as described in Example 1.

7 EXAMPLE 4 This example illustrates the diiference in fluorescence A mixture of 20 ,ul. of ethanolic phenylacetaldehyde (10 mM.), 37.5 pl. of an aqueous solution of each peptide (13.3 mM.), 75 ,ul. of aqueous ninhydrin (30 mM.) and 187.5 ,ul. of phosphate bufier (0.6 M, pH 6.0, 7.0 or 8.0) was incubated for 30 minutes at 60 and diluted with 2.0 ml. of aqueous sodium carbonate (15 mM.). The fluorescence was measured as in Example 1.

incubation at 60 for all pHs.

EXAMPLE 7 This example illustrates the relative fluorescence intensity of some biologically active peptides and amines.

Guanidine 1 Serotonin incubated with the reagents at pH 6.

One m tmole of each compound was used and fluorescence was developed by the procedure described in Example 1 at pH 7. All measurements were made with excitation at 390 my. and emission at 480 mp.

EXAMPLE 8 This example illustrates the difference in sensitivity between the fluorescence assay of the present invention and the standard colorimetric assay.

Absorbance Relative fluorescence Reagent Reagent minus minus Compound Observed blank Observed blank 0. 079 0. 036 254 153 0. 081 0. 038 916 815 0. 083 0. 040 904 803 Glycylglycine 0. 077 O. 034 1, 456 1, 355 Leucylalanine. 0.002 0.049 2,118 2, 017 Tyramine 0. 075 0. 032 1, 128 1,027 Histamine... 0. 070 0. 027 948 847 Blank 0. 043 101 and the possibility of detecting smaller amounts of substrate.

EXAMPLE 9 .Varying amounts of glutamic acid, glycine, leucylalanine and tyramine in 1 ,ul. were spotted on paper or silica gel thin layer plates. After development with solvents and drying, the paper or plate was sprayed with a solution of phenylacetaldehyde (15 mM.) and ninhydrin mM.) in ethanol. The ethanol was allowed to evaporate at room temperature and the chromatogram was then sprayed with 0.2 M phosphate buffer, pH 8.0. The wet chromatogram was then heated at 60 for minutes and after cooling was examined under ultraviolet lamp (366 m With the above method, the detection limit for glutamic acid was 10" mole; for glycine, leucylalanine and tyramine, 10- mole. With the normal Ninhydrin color reaction, the detection limit was 10- mole for all four.

EXAMPLE 10 Ten #1. of an amino acid standard solution (containing l mole/ml each of common amino acids and ammonia) was applied to a 0.9 cm. x 55 cm. column of 7.5% cross-linked sulfonated styrene copolymer resin (Beckman UR- resin for the separation of acidic and neutral amino acids) and developed at 70 ml./hr., first with 117 ml. 0.2 N sodium citrate, pH 3.25, then with 140 ml. 0.2 N sodium citrate, pH 4.3. The starting temperature was and after 30 minutes it was raised over a 1 hour-period to and held at this temperature throughout the remainder of the experiment. The efiluent from the column was split into two streams in the ratio of 9: 1. The larger of the streams was passed through a Beckman Model 120-'C amino acid Analyzer in the normal fashion. The smaller stream, flowing at 0.1 ml./min., was mixed with 0.3 mL/min. of a stream of a solution prepared by mixing 2.67 g. ninhydrin, 180 mg. phenylacetaldehyde, 300 ml. of ethanol and 150 ml. water, and a stream of 0.2 M sodium phosphate buffer, pH 7.5, flowing at 2.0 mL/min. These flow rates were controlled by means of a proportionating pump. The total flow rate after mixture was 2.4 ml./min. An air bubble was introduced approximately every 1.5 seconds to separate the stream into small segments. The flowing stream was passed through a short mixing coil and then into a coil inserted in a heating bath maintained at The total time for the stream to pass through this heating coil was 30 minutes. After coming out of the heating coil, the stream was cooled to 17 C. and introduced into a 1 cm. Aminco half cell provided with a debubbler. Seventy-five percent of the flow passed through the cell (1.8 mL/min.) and 25% was removed in the debubbler. The flow cell was contained in an Aminco Fluoro-Microphotometer equipped with an watt General Electric mercury vapor lamp, an Aminco No. 7.51 filter for excitation, an aperture plate set at maximum excitation and an Aminco No. 4 filter for detection. The fluorophotometer was operated at a high voltage setting of 40, a sensitivity setting of 50% and an attenuation setting of 3% full scale (corresponding to 3 mv. for full scale reading). The output of the fiuorophotometer was coupled to a Beckman 10 inch recorder with a chart speed of 0.1 inch/min.

The recorder used in the Beckman Amino Acid Analyzer for the normal colorimetric analysis was operated at a 5 mv. full scale sensitivity and recorded the normal 570 m output of the colorimeter. The chart speed was 0.1 inch/min.

A parallel experiment was run using a 0.9 cm. X 5 cm. column of 8% cross-linked sulfonated styrene copolymer resin (Beckman PA-35 resin for separating basic amino acids).

The attached drawing shows the recorder tracings made under the above-described conditions. The first run (shown at the left) is for the basic amino acids using the PA-35 column. The second run is for the neutral and acidic amino acids using the UR-3O column.

The chart below presents the data of the attached drawing in tabular form. The relative areas under each peak recorded by both the colorimetric and fiuorometric methods is presented. These areas have been corrected for differences in the amount of sample being measured by each method and difference .in the attenuation of the recorders used. The figures as presented represent the integrated response of each detector under identical conditions produced by equal amounts of each amino acid.

Relative area under peaks of drawingco1:rected Amino acid Colorimetric Fluorometric oH lysine 252 900 Lysine 150 760 H1st1d1ne 1, 350 Ammonia 49 0 Argmrne. 106 705 Cystelc acid 114 430 Aspartic acid- 145 260 Threonine 179 1, Ser1ne 270 2, 960 Glutamic acid 190 865 Glyc ne 152 2, 370 Alanine 136 755 Cystine 161 1,500 Valino. 132 935 Niethionine 157 1, 840 Norleucine 145 1,090 Isoleucine 125 1, 760 Leucine. 144 1,750 Tyrosine 1, 370 Phenylalanine 154 1, fi-Alanine 100 2,120

I claim:

1. A method for determining a primary amine-containing compound in a mixture containing same which method comprises contacting said mixture with ninhydrin and an aryl alkyl aldehyde selected from the group consisting of phenylacetaldehyde and 4-methylphenylacetaldehyde heating the above treated mixture to produce a fluorescent substance therein fluorescing said substance and detecting the fluorescence.

2. The method of claim 1 wherein said treatment is done in solution.

3. The method of claim 2 wherein the aryl alkyl aldehyde is phenylacetaldehyde.

4. The method of claim 2 wherein the pH of the solution is between 6 and 8.

5. The method of claim 2 wherein the molar amounts of ninhydrin and aryl alkyl aldehyde are in excess as compared with the molar amount of primary amine-containing compound.

6. The method of claim 2 wherein the primary aminecontaining compound is an ot-amino acid.

7. The method of claim 2 wherein a fluorophotometer is utilized for fluorescing said substance.

8. The method of claim 2 wherein the concentration in solution of a known primary amine-containing compound is quantitatively determined by (a) determining the fluorescence produced by a measured concentration of said amine, (b) determining the fluorescence produced by the unknown concentration of said amine, and (c) obtaining the unknown concentration by multiplying the known concentration by the ratio of the unknown to the known fluorescence values.

9. The method of claim 2 wherein the primary aminecontaining compound is a polypeptide.

10. The method of claim 9 wherein the polypeptide is selectively determined in a pH range of 6.0 to 7.0.

11. The method of claim 1 wherein the primary aminecontaining compound is present on at least a portion of a surface of a paper or thin-layer chromatographic medium and the ninhydrin and aryl alkyl aldehyde are applied to said surface of said chromatographic medium.

12. The method of claim 11 wherein the aryl alkyl aldehyde is phenylacetaldehyde.

13. The method of claim 11 wherein the pH of the chromatographic medium is kept between 6 and 8.

14. The method of claim 11 wherein the molar amounts of ninhydrin and aryl alkyl aldehyde are in excess as compared with the molar amount of the primary amine-containing compound.

15. The method of claim 14 wherein the aryl alkyl aldehyde is present in a 3-fold molar exces as compared with the ninhydrin.

16. The method of claim 14 wherein the primary aminecontaining compound is an u-amino acid.

17. The method of claim 14 wherein the primary aminecontaining compound is a polypeptide.

18. The method of claim 1 wherein the primary aminecontaining compound is determined by an automated procedure which comprises (a) continuously contacting a sample stream containing said primary amine-containing compound in solution with a fixed proportion of ninhydrin and aryl alkyl aldehyde, (b) continuously passing the mixture obtained in step (a) through a heating zone to produce a stream containing a fluorescent substance, (c) passing the solution obtained in step (b) at constant rate through a flow cell of a fluorophotometer whereby any fluorescent material present is fiuoresced and detected.

19. The method of claim 18 wherein the sample stream is at least a portion of the eflluent of a chromatographic column.

20. The method of claim 18 wherein the pH of the solution being fluoresced is maintained between 6 and 8.

21. The method of claim 18 wherein the primary aminecontaining compound is an a-amino acid.

22. The method of claim 18 wherein the primary aminecontaining compound is a polypeptide.

23. The method of claim 18 wherein the total amount of a specific known primary amine-containing compound present in the sample stream is quantitatively determined by (a) determining the total fluorescence produced by a known quantity of said primary amine-containing compound, (b) determining the total fluorescence produced by the unknown quantity of said primary amine-containing compound under the same conditions as step (a), and (c) obtaining the unknown amount by multiplying the known quantity by the ratio of the unknown to the known total fluorescence.

24. The method of claim 18 wherein the aryl alkyl aldehyde is phenylacetaldehyde.

25. The method of claim 24 wherein the molar amounts of ninhydrin and phenylacetaldehyde are in excess as compared with the molar amount of primary amine-containing compound.

26. A reagent for the determination of primary aminecontaining compounds consisting essentially of a solution of ninhydrin and an aryl alkyl aldehyde selected from the group consisting of phenylacetaldehyde and 4-methylphenylacetaldehyde in an inert organic solvent.

27. The reagent of claim 26 wherein the aryl alkyl aldehyde is phenyl acetaldehyde.

28. The reagent of claim 27 wherein the ninhydrin is present in about a 10-fold molar excess as compared with the phenylacetaldehyde.

29. The reagent of claim 27 wherein the phenylacetaldehyde is present in about a 3-fold molar excess as compared with the ninhydrin.

References Cited UNITED STATES PATENTS 3,306,096 2/1967 Hana et al. 7323.1 3,334,969 8/1967 Catravas 23-230 R 3,506,824 4/1970 Beroza et a1 250-72 R X MORRIS O. WOLK, Primary Examiner R. M. REESE, Assistant Examiner U.S. Cl. X.R. 

